Bioinspired Strategy for the Ribosomal Synthesis of Thioether-Bridged Macrocyclic Peptides in BacteriaNina Bionda, Abby L. Cryan, and Rudi Fasan*

Department of Chemistry, University of Rochester, Rochester, New York 14627, United States

*S Supporting Information

ABSTRACT: Inspired by the biosynthetic logic of lanthipeptide natural products,a new methodology was developed to direct the ribosomal synthesis of macrocyclicpeptides constrained by an intramolecular thioether bond. As a first step, a robustand versatile strategy was implemented to enable the cyclization of ribosomallyderived peptide sequences via a chemoselective reaction between a geneticallyencoded cysteine and a cysteine-reactive unnatural amino acid (O-(2-bromoethyl)-tyrosine). Combination of this approach with intein-catalyzed protein splicingfurnished an efficient route to achieve the spontaneous, post-translational formationof structurally diverse macrocyclic peptides in bacterial cells. The present peptidecyclization strategy was also found to be amenable to integration with split intein-mediated circular ligation, resulting in theintracellular synthesis of conformationally constrained peptides featuring a bicyclic architecture.

Methods for generating macrocyclic peptides, andcombinatorial libraries thereof, have attracted consid-

erable interest owing to the peculiar conformational andmolecular recognition properties of this structural class andtheir promise toward addressing challenging drug targets.1,2 Tothis end, one approach has involved the generation andmanipulation of natural cyclopeptide scaffolds via thereconstruction and engineering of their respective biosyntheticpathways.3−7 Following an alternative approach, other groups,including ours, have focused on implementing strategies toenable the macrocyclization of ribosomally derived polypep-tides of arbitrary sequence.8,9 Particularly attractive features ofthe latter are their high combinatorial potential and thepossibility to interface the resulting libraries of constrainedpeptides with powerful display platforms. Despite significantcontributions in this area,10−18 these approaches have yetremained largely limited to the production of macrocyclicpeptides in vitro.8,9 As a notable exception, there is the splitintein-mediated circular ligation method (SICLOPPS) intro-duced by Benkovic and co-workers,19 in which head-to-tailcyclic peptides are generated via circularization of an internalpeptide sequence upon a trans splicing reaction involvingflanking domains from the natural split intein DnaE(Supplementary Figure S1). Enabling the synthesis of cyclicpeptides in living cells, this approach has provided a powerfultool for the discovery of cyclopeptide inhibitors against avariety of protein targets upon integration with an intracellularselection or reporter system.20−22 A limitation of this methodhowever is the accessibility of a single type of cyclic peptidetopology (i.e., N-to-C-end cyclic peptide). Furthermore,cyclization efficiency is largely affected by the composition ofthe target peptide sequence.22−24 Thus, more general andversatile methods to direct the ribosomal synthesis ofmacrocyclic peptides would be highly desirable in order toexpand current capabilities toward exploring and exploiting

peptide macrocycles for drug discovery and chemical biologyapplications.To this end, we developed and report here an efficient

strategy for the production of structurally diverse thioether-linked macrocyclic peptides in living bacterial cells (E. coli).Inspiration for this approach was drawn from the ‘logic’underlying the biosynthesis of lanthipeptides, a class ofribosomally derived polycyclic peptides constrained by intra-molecular thioether bonds.25,26 As schematically illustrated inFigure 1A, these natural products are initially produced as linearprecursor polypeptides via ribosomal synthesis. Recognition ofthe N-terminal leader sequence by dehydratase enzymes thenmediates the conversion of Ser and Thr residues located withinthe core peptide into dehydroalanine (Dha) and dehydrobu-tyrine (Dhb), respectively. The signature thioether cross-linksare subsequently formed via enzyme-assisted Michael additionof cysteine sulfhydryl groups onto these electrophilic α,β-unsaturated amino acid residues. Finally, removal of the leaderpeptide by a downstream protease releases the maturelanthipeptide.25,26

Inspired by this reaction sequence, we sought to develop astrategy for the ribosomal generation of conformationallyconstrained peptides through the combination of peptidecyclization via an inter-side-chain thioether linkage withproteolytic release of the resulting macrocyclic peptide.Conceivably, achieving this goal in an enzyme-independentmanner would involve the challenge of building in thestructural elements necessary for promoting these trans-formations directly into the genetically encoded, ribosomallyproduced precursor polypeptide. As outlined in Figure 1A, we

Received: April 26, 2014Accepted: July 31, 2014Published: July 31, 2014

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reasoned this task could be achieved via (a) a chemoselectivereaction between a cysteine and a ribosomally incorporatedunnatural amino acid bearing a cysteine-reactive side-chaingroup and (b) spontaneous release of the macrocyclic peptideby means of an intein-based protein splicing element.To implement this idea, we envisioned that the target

unnatural amino acid (UAA) ought to satisfy several criteria.First, its side-chain electrophilic group should be sufficientlyreactive to enable efficient peptide cyclization upon reactionwith a proximal cysteine in intramolecular settings, but not too

reactive in order to avoid side reactions with competingnucleophiles present in the cellular environment (e.g.,glutathione). In addition, it should be amenable to proteinincorporation via an aminoacyl-tRNA synthetase (AARS)/tRNA pair with orthogonal reactivity.27

With these considerations in mind, we decided that O-(2-bromoethyl)-tyrosine (O2beY, Figure 1B) could meet theaforementioned requirements, as suggested by its sluggishreactivity in the cyclization of cysteine-containing peptides invitro15 and its structural similarity to O-propargyl-tyrosine(OpgY), for which an amber suppressor AARS/tRNA pair wasmade available.28 Accordingly, the envisioned approach wouldentail cyclization of a precursor polypeptide via a thioetherbond-forming reaction between O2beY and a proximalcysteine, followed by intein-mediated release of the macrocyclicpeptide (Figure 1B).To evaluate the feasibility of this design, a first series of

constructs was utilized (entries 1−9, Figure 2A), which encodefor 12- to 16-amino acid long target peptide sequences and inwhich the O2beY and Cys residues are spaced from each other

Figure 1. (A) Schematic representation of lanthipeptide biosynthesis(i) and our bioinspired strategy for ribosomal synthesis of thioether-bridged macrocyclic peptides (ii) and bicyclic peptides (iii). (B)Strategy for ribosomal synthesis of thioether-bridged macrocyclicpeptides. The linear precursor polypeptide comprises an N-terminaltail (pink), the unnatural amino acid O-(2-bromoethyl)-tyrosine(O2beY), a variable target sequence (green) containing the reactivecysteine (purple), and GyrA intein (blue). Depending on the nature ofthe “I−1” residue (orange), the macrocyclic peptide can be released invitro (path A) or directly in vivo (path B).

Figure 2. (A) Protein constructs used in this study. Notes: aTheresidues involved in the thioether linkage are highlighted in green(O2beY) and red (Cys). Initial methionine is omitted. bRelativeposition of the cysteine with respect to the unnatural amino acidO2beY ( = ‘Z’). (B) Dependence of macrocyclization efficiency on therelative position of the Cys residue with respect to the unnaturalamino acid O2beY (‘Z’). Proteins were isolated after expression for 12h at 27 °C (see Supporting Information for details).

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by an increasing number of intervening residues (i.e., from Z+1to Z+12). In the respective genes, an amber stop codon (TAG)was introduced after the initial Met-Gly to allow for the site-specific incorporation of O2beY into the target sequence. Thelatter was then genetically fused to an engineered variant ofMxe GyrA intein, whose C-terminal Asn198 was mutated to Alato abolish its self-splicing activity while preserving its ability toform a thioester bond at the N-terminal end via N → S acyltransfer (dotted box, Figure 1B).To identify a viable AARS/tRNA pair for O2beY

incorporation into these constructs, we initially tested theMethanocaldococcus jannaschii tyrosyl-tRNA synthetase variantpreviously evolved for recognition of the structurally relatedOpgY (OpgY-RS).28 This choice was motivated based on the‘polyspecificity’ often exhibited by orthogonal AARSs towardrelated UAA structures.29,30 Gratifyingly, using OpgY-RS,O2beY could be successfully incorporated into a model proteinconsisting of Yellow Fluorescent Protein (YFP) with an N-terminal amber stop codon (Figure 3B). To improve its

efficiency toward O2beY incorporation, OpgY-RS was sub-jected to further mutagenesis. To this end, an homology modelof this enzyme was first generated on the basis of the availablecrystal structure of MjTyr-RS in complex with its nativesubstrate, tyrosine.31 Then, the unnatural amino acid O2beYwas docked into the enzyme active site (Figure 3A). Inspectionof the model suggested that an Ala32Gly mutation wouldexpand the active site cavity to better accommodate the 2-bromo-ethoxy group in O2beY. Rewardingly, the resultingAARS variant (called O2beY-RS) was found to enable theribosomal incorporation of O2beY with significantly higherefficiency compared to OpgY-RS, while maintaining discrim-inating selectivity against the natural amino acids (Figure 3B).Comparison of the expression yields of YFP(O2beY) versuswild-type YFP indicated that the efficiency of amber stop codonsuppression with O2beY-RS was excellent (85%).

With a suitable AARS/tRNA pair for O2beY incorporation inhand, the constructs corresponding to entries 1−9 (Figure 2A)were produced in E. coli BL21(DE3) cells using a dual plasmidsystem (see Supporting Information for details). To betterexamine both the occurrence and efficiency of the thioetherbond-forming reaction according to the strategy of Figure 1B,the aforementioned constructs were designed to contain a Thrresidue at the position preceding the intein (‘I−1 site’). Thissubstitution minimizes premature hydrolysis of GyrA-fusionproteins during expression,14,32 thereby facilitating analysis ofthe target peptide sequences after chemically induced splicingof the intein from the purified proteins in vitro (Figure 1B, pathA). This procedure would also permit the isolation of anyproduct resulting from the unselective reaction of O2beY withother nucleophiles in vivo. Accordingly, after purification, theproteins were treated with benzyl mercaptan to release the N-terminal peptides. The reaction mixtures were then analyzed byLC−MS to detect and quantify the amount of the desiredthioether-linked macrocyclic product as well as that of theuncyclized linear peptide, as judged on the basis of the peakareas in the corresponding extracted-ion chromatograms (seeFigure 4A and Supplementary Figures S3−S10). As summar-ized in Figure 2B, these studies revealed that the macro-cyclization had occurred with very high efficiency (80−95%)across the constructs with Cys and O2beY being separated byone (Z+2) to seven (Z+8) residues. Increasing this distance(i.e., Cys at Z+10 and Z+12, entries 8 and 9 in Figure 2A)resulted in a noticeable increase of the acyclic product (50−80%, Figure 2B), thus defining the upper limits for themacrocycle size accessible through this method. Interestingly,when the Cys was located immediately adjacent to theunnatural amino acid (entry 1, Figure 2A), minimal cyclization(5%) was observed. A similar lack of reactivity was observed bySuga and co-workers in the context of in vitro translatedpeptides containing a cysteine-reactive N-terminal 2-chloroa-cetyl moiety,33 and this result can be rationalized here on thebasis of the unfavorable 14-membered macrocycle formedwhen the O2beY/Cys pair are in a i/i+1 relationship. For eachconstruct tested, the identity of the macrocyclic product couldbe further confirmed by analysis of the corresponding MS/MSfragmentation spectrum as illustrated in Supplementary FigureS2.Importantly, GyrA intein contains a Cys at its N-terminal

end, which is crucial for mediating protein splicing in thecontext of our planned strategy for producing these peptidemacrocycles inside the cells (Figure 1B). Since this residue ispartially buried within the active site,34 we did not expect it toreadily react with the O2beY side-chain. Notably, quantitativesplicing of the GyrA moiety upon treatment of all thesecontructs with benzyl mercaptan (Figure 4A and Supplemen-tary Figure S17) indicated that no reaction occurred betweenO2beY and the catalytic Cys at the intein I+1 site. Furthermore,no adducts or dimers were observed for any of the constructsdescribed above, including those undergoing only partialcyclization (i.e., entries 8 and 9, Figure 2A). Altogether, theseresults evidenced the high chemo- and regioselectivity of themacrocyclization reaction.In the interest of determining whether the thioether bond-

forming reactivity is preserved if the order of Cys and O2beY isreversed, the two constructs corresponding to entries 10 and 11in Figure 2A were prepared. Here, the reactive Cys is locatedupstream of the unnatural amino acid and specifically atpositions Z-6 and Z-8. Analysis of these constructs according to

Figure 3. Aminoacyl-tRNA synthetase for O2beY incorporation. (A)Model of OpgY-RS active site as generated based on the crystalstructure of MjTyr-RS in complex with tyrosine (PDB code 1J1U).The bound substrate (Tyr, yellow), docked O2beY (green), andmutated acid residues (red) are highlighted. The van der Waals sphereof the Ala32 residue is shown as a dotted sphere. (B) YFP-basedscreen indicating the relative efficiency of OpgY and O2beYincorporation via amber stop codon suppression with OpgY-RS andO2beY-RS. (C) MS spectrum of YFP protein incorporating O2beY(YFP(O2beY)).

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the procedure described above (Supplementary Figures S11and S12) revealed the occurrence of the desired cyclic peptideas the largely predominant product (>99%), i.e., withcomparable (Z-6) or even higher (Z-8) efficiency than thecorresponding Z+6 and Z+8 counterparts (Figure 2B). Thesedata clearly showed that either arrangement of the Cys/O2beYpair within the target sequence is compatible with macro-cylization.Having established the versatility of the approach toward

obtaining structurally diverse macrocyclic peptides either linkedto the N-terminus of a protein or in isolated form after inteinsplicing in vitro, we next investigated whether this strategycould be further evolved to permit the production ofmacrocyclic peptides in vivo. In previous studies,18 weestablished that certain amino acid substitutions at the levelof the I−1 site, and in particular Asp and Lys, can stronglypromote N-terminal splicing of GyrA intein during recombi-nant expression. This effect is likely due to the ability of theseresidues to favor hydrolysis of the intein-catalyzed thioesterlinkage through their nucleophilic side-chain groups. Whileundesirable in the context of our MOrPH synthesis method-ologies,18 we envisioned this reactivity could be leveraged herefor mediating the spontaneous release of the macrocyclicpeptide from the precursor protein as outlined in Figure 1B(path B). To test this idea, the constructs corresponding toentries 12 and 13 in Figure 2A were generated. In addition toan Asp residue at the I−1 site, these contructs were designed to

encompass the sequence of two streptavidin-binding peptidespreviously isolated by phage display35,36 as a way to facilitatethe isolation of the target macrocyclic peptides from the cells.Accordingly, after expression of these constructs in E. coli, cellswere lysed and the cell lysates were passed over streptavidin-coated agarose beads. To our delight, LC−MS analysis of theeluates revealed the occurrence of the expected peptidemacrocycles, as illustrated by the chromatograms and MS/MS spectra in Figure 4B and Supplementary Figures S13 andS14. Since the uncyclized peptide could also be capturedthrough this procedure, these analyses also showed that thedesired macrocyclic product was formed with high efficiency ineach case (i.e., >95% for Strep1-Z5C; 70% for Strep2-Z7C).Furthermore, both precursor polypeptides were found to haveundergone complete splicing in vivo (Figure 4B andSupplementary Figure S18A,B). Since O2beY-mediatedalkylation of the intein catalytic cysteine would prevent proteinsplicing, the latter results further higlighted the high degree ofregioselectivity of the macrocyclization reaction. Collectively,the results obtained with the streptavidin-binding sequences ofconstructs Strep1-Z5C and Strep2-Z7C provide a proof-of-principle demonstration of the feasibility and efficiency of thestrategy of Figure 1B for directing the synthesis of cyclo-peptides in living bacterial cells. Interestingly, the cyclizationyield observed with these sequences correlated very well withthe reactivity trend measured across the previous contructs(Figure 2B), suggesting that this parameter is rather predictable

Figure 4. Chemical structure (top), LC−MS extracted-ion chromatogram (center left), and MS/MS fragmentation spectrum (bottom) ofrepresentative macrocyclic peptides prepared from construct 12mer-Z6C (A), Strep1-Z5C (B), and cStrep3(C)-Z3C (C). MS spectra of theprecursor proteins after affinity purification and thiol-induced splicing (12mer-Z6C) and directly from cell lysate (Strep1-Z5C and cStrep3(C)-Z3C)are also shown (center right). Data for the other constructs can be found in Supplementary Figures S2−S19.

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on the sole basis of the Cys/O2beY distance and despite thedifference in the composition of the target peptide sequence.These positive results prompted us to test whether our

bioinspired approach could be further extended to enable theribosomal synthesis of bicylic peptides via the integration ofO2beY/Cys-mediated macrocyclization with split intein-cata-lyzed circular ligation.19 If viable, this second strategy (Figure1A(iii)) could provide the complementary capability ofgenerating macrocyclic peptides that are constrained bymeans of an N-to-C-end cyclic backbone and an intramolecularinter-side-chain thioether linkage. Implementation of thisdesign presented the challenge that two cysteine residues areinvolved in the trans splicing process leading to the head-to-tailcyclopeptide (referred to as IntC+1 and IntN+1 cysteine; seeSupplementary Figure S1), which could potentially cross-reactwith O2beY. However, on the basis of the reactivity studiesdescribed in Figure 2B, we envisioned this challenge could betackled by placing the unnatural amino acid in i/i+1arrangement with respect to the IntC+1 cysteine and by placingthe reactive cysteine at a closer distance to O2beY compared tothe IntN+1 cysteine, as schematically outlined in Figure 5.

According to these design principles, the cStrep3(C)-Z3Cconstruct was prepared (entry 14, Figure 2A). A relatedconstruct where the IntC+1 cysteine is replaced with serine(entry 15, Figure 2A) was also prepared, as this substitution iscompatible with split intein-catalyzed peptide cyclization.23 Ineach case, the target peptide sequences were designed on thebasis of a previously described HPQ motif-containing cyclo-peptide capable of binding streptavidin37 in order to facilitateisolation of the peptide products from the cells. Lysates from E.coli cells expressing these constructs were processed asdescribed above for the other streptavidin-binding peptides.Notably, the desired bicyclic peptide was isolated as the largelypredominant product in both cases (>95%), as determined byLC−MS. The bicyclic structure of these compounds wasfurther evidenced by the corresponding MS/MS fragmentationspectra (Figure 4C and Supplementary Figures S15 and S16).Treatment of the bicyclic peptides with the thiol-alkylatingiodoacetamide resulted in a 57 Da increase in molecular massand shift of the peptide retention time for the product of thecStrep3(C)-Z3C precursor protein but not for that ofcStrep3(S)-Z3C, which is consistent with the presence of afree thiol from (from IntC+1 cysteine) in the former but not inthe latter. To allow measurement of the extent of post-translational self-processing of these precursor polypeptides invivo, a chitin-binding domain was included at the C-terminus of

the IntN domain (Figure 2A). LC−MS analysis of the proteinfraction eluted from chitin beads showed that the split intein-mediated cyclization has occurred quantitatively with bothcStrep3(C)-Z3C (Figure 4C) and its serine-containing counter-part (Supplementary Figure S18D). Altogether, these resultsdemonstrate the possibility of integrating our peptidecyclization strategy with split intein-mediated protein circular-ization to enable the formation of bicyclic peptides in E. coli.In conclusion, we have developed two complementary,

versatile methodologies to direct the production of ‘naturalproduct-like’ macrocyclic peptides constrained by an intra-molecular thioether bridge in bacterial cells. A key feature ofthese methods is that the structural elements and reactivitydriving the peptide macrocyclization process are built into thegenetically encoded polypeptide precursor. Using this ap-proach, peptide macrocycles of various ring size andcomposition could be prepared efficiently and with predictableregioselectivity. Importantly, the possibility to generate thesemacrocyclic peptides in protein-fused or isolated form make thepresent methodologies directly amenable to integration withwell-established display platforms (e.g., phage38 or mRNAdisplay39) or intracellular selection systems,20,21 respectively,for library screening. We also anticipate that the strategiesoutlined in Figures 1B and 5 can be of rather general value, thatis, applicable to other cysteine-reactive unnatural amino acids.Work in this direction is currently ongoing in our group, whichincludes exploring the possibility of extending this approach tothe synthesis of conformationally constrained peptides ineukaryotic cells.40

■ ASSOCIATED CONTENT*S Supporting InformationDetailed experimental procedures, synthetic procedures(O2beY synthesis), additional LC−MS chromatograms, andMS/MS and MS spectra analysis of the macrocyclic products.This material is available free of charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: fasan@chem.rochester.edu.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported in part by the U.S. National ScienceFoundation grant CHE-1112342 and in part by the NationalInstitute of Health grant R21CA187502. MS instrumentationwas supported by the U.S. National Science Foundation grantsCHE-0840410 and CHE-0946653. The authors are grateful toProf. P. G. Schultz (TSRI) for providing the pEVOL vector.

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Figure 5. Schematic representation of the strategy for in vivo synthesisof bicyclic peptides via combination of O2beY/Cys cyclization andsplit intein-mediated circular ligation.

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